The aim of this program project is to integrate the fields of cell biology, biochemistry, fluorescent probe chemistry, biophysics, and computer science for the study of cell motility and endocytosis. The focus of the approaches is on the application of fluorescence techniques to define the temporal and spatial dynamics of selected cellular physiological events. Fluorescent analogs of specific contractile proteins will be incorporated into living cells and analyzed by digital image analysis, fluorescence recovery after photobleaching, flow cytometry, total internal reflection fluorescence and by standing-wave luminescence microscopy. The processing of ligands through the various stages of endocytosis will be studied by fractionating cells and analyzing the endosomes and various other organelles by flow cytometry. New fluorogenic substrates of specific enzymes, including lysosomal enzymes, will be prepared to study the processing of ligands temporally and spatically. These experiments will be performed on mammalian cells in culture as well as human leukocytes. The projects should help to define the molecular events involved in cell movements and the pathways of processing extracellular ligands starting from the binding of the ligand to the cell surface.